When conducting genetic manipulations or other treatments of cells or tissue, it is often necessary to partially or completely penetrate the cell walls and/or membranes with a biological or other agent. This penetration is necessary in order to achieve a desired effect on the cell wall and/or internal cellular elements such as the cytoplasm, nucleus, plastids, chromosomes, plasmids, etc. The objective of such a procedure may be, for example, the destruction of selected substances or the production of new or improved biological characteristics. These procedures can be used to modify a plant, animal, or microbe to improve, for example, growth rate, disease resistance, or protein production. Other applications include the tagging of cells for tracking and identification, or the micromanipulation of cells by in situ rotation or displacement in space.
In genetic research, for example, such methods are used to penetrate tissue and cells with particles precoated with DNA encoding genes of interest; cell penetration is followed by DNA delivery into the cell nucleus or organelle. To reach the intracellular space and then the cell subcellular structure, the particles must traverse formidable cell walls and membranes. Because these cell walls are difficult to penetrate, the particles carrying the DNA are driven into the cells by the force of an explosive or an electrical discharge so that the kinetically driven particles smash into the target tissue. Even then, in order to have the necessary kinetic energy for penetration of certain targets and to certain depths, the particles must be several micrometers in diameter. Thus, the implantation process results in appreciable cell damage due to the impact of the particles and/or due to sonic concussion from the particle-propelling discharge. Some cell tissue, drawing upon its natural strength, may recover from this trauma sufficiently to integrate the newly delivered genetic material into its chromosomes; however, a significant percentage of the tissue is unable to do so.
These prior methods of delivering particles to cells also lack sufficient control over particle size distribution, particle coating quality, and the velocity and direction of travel of the particles, resulting in a lack of predictability and reproducibility of the particle delivery technique. The prior delivery techniques are further disadvantaged because they require that the target tissue be maintained in a vacuum which removes moisture from the treated tissue contributing to tissue degradation. Moreover, the apparatus for performing the implantations requires time-consuming set up prior to each implantation cycle and is cumbersome to clean after same so that the throughputs of the apparatus are relatively low.
Other methods employed or suggested for direct gene delivery to cells include the use of microlasers, microbead vortexing, electrofusion, chemical fusion, microinjection, and electroporation. Such techniques all rely on increasing the permeability of the tissue cells by physically, chemically, or electrically disrupting cell walls and/or membranes temporarily; exogenously added DNA may then enter the cell through the temporary ruptures. Some of these methods, including microinjection and fusion of preselected protoplasts or subprotoplasts, require working at the single cell level. This necessitates micromanipulation of the cells, often involving immobilization by agarose plating or pipette suction. Such micromanipulations must be carried out with a microscope placed in the sterile environment of a laminar flow hood, which can be very cumbersome. Also, controlled fusion, for example in the production of somatic hybrids, requires bringing the fusion partners into close proximity which is technically difficult to accomplish. Another bottleneck in plant and other genetic transformation systems is the relative inefficiency of selection following gene transfer. A means to enrich for penetrated cells or organelles prior to, or in place of, selection by use of antibiotics, herbicides, osmotics or toxins would greatly improve the efficiency of a transgenesis system.
A number of magneto-mechanical systems have recently been devised whose purpose is to deliver certain reactive substances to a target site using sharp-tipped microparticles as carriers which penetrate the target sites such as pollen, cells, organisms, etc. to deliver these substances in singular or multiple sequential entries by desorption from the microparticles to cause a change in the target site, such as altering a genetic trait or curing a disease.
U.S. Pat. No. 5,516,670 discloses a method for delivering particles into cellular specimens by means of a non-uniform, convergent magnetic field and is incorporated herein by reference.
There still exists a need for particles which are designed to effectively perform each of a variety of tasks. For example, particles can serve as xe2x80x9cpayload carriersxe2x80x9d such as to either deliver or extract one or more substances of interest to or from target sites. In another example, particles can xe2x80x9cmarkxe2x80x9d the target for detection and/or counting purposes. Particles themselves may be the therapeutic agent, as in heat therapy. In addition, the subject particles can be furnished with properties which enable, or enhance, chemical interactions to achieve the desired treatment goals. Because of the variety of treatments required such as genetic alteration of a cell via DNA coupling or gene splicing or addressing different target sites such as pollen, cells, meristems, or human cancers, tumors, lymph nodes or nerve endings, a wide variety of microparticles are needed in terms of length, width, tips, geometries, and basic materials.
The subject invention provides methods and apparatuses for the manufacture of magnetizable carrier particles (xe2x80x9cmicromagnetsxe2x80x9d). In addition, the subject invention pertains to particles having one or more of a variety of particle configurations and/or functional features. These particle configurations and/or functional features can be tailored to achieve one or more desired missions. The subject invention also pertains to methods and apparatuses for the delivery of particles to target materials, in order to accomplish one or more of a variety of missions.
In a specific embodiment of the subject invention, acicular and other particles with a lengthwise dimension that are uniform and homogenous in their geometry are manufactured and provided with magnetizations. In this way, predictable mechanical force responsivity can be achieved when these particles are subjected to an external magnetic field gradient. Preferably, saturation magnetization can be provided to the particles in order to yield optimal force for a particular particle size when inserted into a magnetic field gradient.
In another embodiment of the subject invention, the size, length, cross-sectional area, and/or shape and geometric contours of the particles, including asymmetry of mass, are selected such that the particle is optimally related to the target body. Accordingly, the particle""s configuration can vary based on the target""s size, shape, resistance against penetration, and other characteristics of the particle. For example, the subject particles can be provided with sharp xe2x80x9ctipsxe2x80x9d at one or both ends. These sharp tips enable the particles to exert powerful pressures (force per unit area) at the point of contact between particle tip and target body, enhancing the ability of the particles to breach a protective wall of the target and to enter its interior space. One such embodiment involves providing a particle with a sharp xe2x80x9ctipxe2x80x9d at one end and a broader, nail-head-like tail at the other end, such as to enable partial penetration of the particle and, if desired, subsequent retraction of the particle. Such a particle can be retracted using, for example, a tape-like backing which can adhere to the particle""s broader tail.
In another specific embodiment, the surface of the particles can be shaped such as to attract and/or accommodate molecular matter (substances of interest). Examples of such molecular matter include DNA, RNA, proteins, cell material, salts, sulfonamides, pharmaceutical substances, enzymes, viruses, and marker substances like upconverting phosphors. By shaping the surface of the subject particles to attract and/or accommodate such substances of interest, the subject particles can more effectively deliver this molecular matter to the inside of the target body. The particle can also be designed to enhance detachment of such substances during entry into the target material or after entry into the target material. For example, the molecular matter can detach under the effect of the prevailing force vectors.
In addition, the surfaces of the subject particles can be conditioned such as to more effectively attract and accommodate molecular matter. In specific embodiments, conditioning of the particle surface can encompass altering one or more of the following surface properties of the particle: topography; pH; electrochemical potential; van der Waals forces; surface energy; and other surface conditions. These surface conditions can cause the particle""s surface to readily absorb and hold the payload substances, or to carry precipitated payloads and retain them with less holding force than the desorption or dissolution force is capable of exerting onto the payload. Accordingly, particle surface conditioning can help to assure delivery of the payload substances of interest to the interior cell plasma or other such target body, such as to enable its interaction with said body. Such interaction can include, for example, gene introgression or vaccination with the delivered genetic material.
After the particles have come to rest with respect to the target, for example when an externally applied magnetic field used for propelling the particles is shut off or mechanical or dynamic delivery is terminated, secondary interactions between the particle and body can be accomplished. For instance heating of the particle by activating a resonant circuit loop placed on the particle can trigger the release of material held by the particle, enhance the efficacy of materials released by the particle, and/or heat the target to affect the interaction between the particle and the target. Secondary interaction between the particle and the target can also be enabled by applying an external magnetic field for purposes of, for example, marking, tagging, retrieving, sorting, rotating, counting, and/or detecting the target material.
In order to protect the payloads against shear forces while penetrating the target site and its protective walls, the particles can be encapsulated together with their payloads. Such encapsulation can provide catalyst material for stimulating chemical interactions between payload and target material.
In a further embodiment, the subject particles can be coated by means routine in the art with substances of interest such as materials which attract viruses, bacteria, and/or other organisms or agents. Once attracted, these organisms or agents can be captured on the particle surface. These organisms or agents can then be held to the particles while the particles are, for example, magnetophoretically introduced into or extracted from the target site. These techniques are applicable in vivo and in situ. Accordingly, these techniques can be utilized for installing or removing these entities or other potential malefactors.
Advantageously, the subject particles can, in part due to their small size, shapes, and low surface energy or low coefficient of friction (for example, in an embodiment having a TEFLON coating), readily slip through muscle tissue or other body substances and thereby provide a means for deeply penetrating an entity, such as a human body, without surgery and without inflicting serious injury. The subject technique can be applied to an organ, cell, or other chemical or structural entity whose composition can be altered and remain altered by the presence of the substances of interest delivered to it, as well as the continued presence of the delivered particles until they are removed. The subject particles can initiate a permanent transformation of the consistency and condition of the target site so as to effectuate its healing or its replicative genetic transformation on account of the substances (the payloads) delivered by the carrier particles to the body, cell, pollen, or bioplasma present at the target site. The subject particles can be important elements in a magnetophoretic delivery system, allowing delivery of various substances of interest without the need for surgical invasion or using the blood stream to circulate medicinal substances in order to reach the target site.
The delivery of the subject particles to a target can also be used to initiate a temporary transformation or stimulation either with a payload or alternatively the particles by themselves. For example, they may be employed in order to trigger a cascade of events such as an immune response, a hypersensitive or systemically active resistance response, viral replication or movement, and/or fertilization with extranuclear or satellite genetic material, or particles themselves can be used to cause effects analogous to acupuncture.
Exposure of the subject particles to a preparatory magnetic field prior to executing delivery by magnetophoretic, mechanical, or dynamic means can be used to maintain dispersion, to direct the orientation of, and/or to temporarily immobilize said particles. In a specific embodiment, the subject particles can be coated in a dispersed, oriented, and immobilized state during exposure to a magnetic field. Further immobilization of the oriented, dispersed, and conditioned or coated particles in a matrix or with backing tape suited to the particle delivery method, can be utilized for stabilization of the particles in conjunction with, or in the absence of, a preparatory magnetic field.
Advantageously, the subject particles can be mass produced having various types, sizes, compositions, and features. The subject particles can be manufactured with precision using an apparatus for continuously creating, extracting, gathering, conditioning these particles to accept the substances for which they were designed. The coated particles can then be stored in a suitable medium such that they are ready for use when needed.